Summary/ Abstract p53 is the most commonly mutated tumor suppressor gene in human cancers. Many sites of mutation have been found in >50% of human tumors, in almost every type of cancer. Each p53 mutation confers specific effects on the treatment response through various mechanisms, most of which are unknown. We propose a novel mechanism by which p53 mutations provide a gain of function, namely, resistance to therapy, by inducing the IFN-related DNA damage-resistant signature (IRDS). The IRDS genes are a specific subset of interferon-stimulated genes (ISGs) that are highly upregulated in many types of cancer and that are resistant to DNA-damaging chemotherapy and radiotherapy. Our prior studies showed that persistent simulation of cells with low levels of interferon ? (IFN?) induced the IRDS genes only and increased resistance to DNA damage. In our preliminary study, we found that constitutive expression of IFN? was induced by R175H-p53, one of the most frequently observed p53 mutants, in breast and lung cancer cells. R175H-p53 also increased the expression of IRF9, which enhances resistance to chemotherapy. IRF9 shares a DNA binding domain with IRF3, a major transcription factor that mediates IFN? synthesis. We HYPOTHESIZE that p53 mutations stimulate cancer cells to express low levels of IFN? constitutively by binding of IRF9 to the IFN? promoter, increasing their resistance to DNA damage. In Aim 1, we will test the most frequently found p53 mutations (G245S, R248Q, R248W, R249S, R273H, R273C, R282W) using various types of solid tumor cells. We will elevate the levels of mutant p53, as seen in actual tumors expressing the p53 mutant proteins, and test whether high levels of each mutant p53 increase IFN? expression, as we have already observed for R175H- p53. In Aim 2, we will determine the involvement of IRF9 and STING in IFN? synthesis induced by R175H- p53. We propose here that IRF9, whose expression is increased by R175H-p53, binds to the promoter of the IFN? gene and regulates IFN? expression. We will determine the effect of IRF9 knockdown on R175H-p53- induced IFN? expression and assess IRF9 binding to the IFN? promoter using ChIP. We also propose that IFN? expression is induced by DNA damage, caused by impairment of DNA repair, through activation of STING, a cellular sensor of cytosolic DNA. We will determine the impact of STING phosphorylation by R175H- p53 and test the effect of STING knockdown on R175H-p53-induced IFN? expression in cancer cells. These studies connect two known factors, p53 mutation and IRDS expression, that promote resistance to cancer therapy, proposing a novel mechanism. The knowledge obtained from these studies will enable us to develop new approaches to overcoming resistance to cancer therapy.